Sodium bis[3-[[1-(3-chlorophenyl)-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-4-yl]azo]-4-hydroxy-N-methylbenzene-1-sulphonamidato(2-)]chromate(1-)

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2017-09-25 to 2017-12-15 with the definitive exposure phase from 2017-12-13 to 2017-12-15.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Sodium bis[3-[[1-(3-chlorophenyl)-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-4-yl]azo]-4-hydroxy-N-methylbenzene-1-sulphonamidato(2-)]chromate(1-)

EC Number:

276-067-7

EC Name:

Sodium bis[3-[[1-(3-chlorophenyl)-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-4-yl]azo]-4-hydroxy-N-methylbenzene-1-sulphonamidato(2-)]chromate(1-)

Cas Number:

71839-81-1

Molecular formula:

C34H28Cl2CrN10O8S2.Na

IUPAC Name:

Chromate(1-), bis[3-[2-[1-(3-chlorophenyl)-4,5-dihydro-3-methyl-5-(oxo-kO)-1H-pyrazol-4-yl]diazenyl-kN1]-4-(hydroxy-kO)-N-methylbenzenesulfonamidato(2-)]-, sodium (1:1)

Test material form:

solid: particulate/powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Determination of the test item
All concentration levels and the control were analytically verified via HPLC-DAD in fresh media at the start of exposure (0 hours) and old media at the end of the exposure (48 hours). The method was validated prior to this study according to SANCO 3029/99 rev.4 (2000).

Sampling for the analytical monitoring
At the start of the exposure (0 hours), samples of the freshly prepared test item concentrations were taken and analyzed. At the end of the exposure (48 hours), samples of the old media were taken directly from the test vessels and analyzed.

Range (target)
Recoveries of the test item should be within ± 20% of the nominal concentrations.

Test solutions

Vehicle:

no

Details on test solutions:

Preparation of the stock solution A stock solution of 100 mg/L of the test item was prepared with dilution water two days prior to the start of the exposure (at -48 hours) in a glass flask.
The test item was mixed with the dilution water by constant stirring with a magnetic stirrer at approximately 1100 rpm for 48 hours at room temperature. After completion of stirring, undissolved particles were removed by membrane filtration (membrane filter 0.45 µm, RC, MACHEREY-NAGEL). The filter was saturated in order to avoid adsorption during the filtration. The first 25 mL of the filtrate were discarded. The filtration was interrupted for 15 minutes to allow for adsorption and saturation of the filter material with dissolved test item. Thereafter, the filtration was continued. The next 25 mL were discarded. The following filtrate, i.e. the saturated test item solution, were used as the highest concentration level and as a stock solution for the preparation of further concentration levels by dilution with dilution water. During filtration, the filter was always kept covered.

Test concentrations
Five concentrations of the test item prepared out of the stock solution 100 mg/L in a geometric series with a separation factor of 2.2 by dilution of the stock solution with dilution water, will be tested as follows: 4.27 - 9.39 - 20.7 - 45.5 - 100 mg/L
The test concentrations were selected based on the results of a non-GLP preliminary range finding test and the first definitive test.

Control Dilution water without test item incubated under the same conditions as the test groups

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Test system
Daphnia magna STRAUS (Clone 5).

Reason for the selection of the test system
Daphnia magna is the preferred species in accordance with the test guideline and is bred at the test facility.

Origin
Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), 14195 Berlin, Germany

Breeder
Noack Laboratorien GmbH,
Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany

Culture
In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20  2°C, in an incubator, 16 hours illumination, light intensity of max. 1500 lx

Culture medium
Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L, was used.

Culture feeding
The culture daphnids were fed at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus, with an algae cell density of > 106 cells/mL. The algae were cultured at the test facility.

Origin of the food algae
Sammlung von Algenkulturen (SAG),
Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, 37073 Göttingen, Germany


Composition of the Culture Medium according to ELENDT (1990)
Component Concentration [mg/L]
CaCl2 x 2 H2O 147
MgSO4 x 7 H2O 123
KCl 5.80
NaHCO3 64.8
Na2SiO3 4.30
NaNO3 0.27
KH2PO4 0.14
K2HPO4 0.18
Na2EDTA x 2 H2O 5.00
FeSO4 x 7 H2O 1.99
H3BO3 0.29
MnCl2 x 4 H2O 0.36
LiCl 0.30
SrCl2 x 6 H2O 0.15
RbCl 0.071
NaBr 0.016
Na2MoO4 x 2 H2O 0.063
CuCl x 2 H2O 0.017
ZnCl2 0.013
CoCl2 x 6 H2O 0.010
KJ 0.00325
Na2SeO3 0.00219
NH4VO3 0.000575
Thiaminhydrochloride 0.075
Cyanocobalamine 0.0010
Biotin 0.00075
pH 8.2  0.8

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

Dilution water
0 hours:Total hardness [mg CaCO3/L]: 175

Test temperature:

18 - 22°C, constant within ± 1°C
19.0 °C

pH:

see any other information on materials and methods incl. tables

Dissolved oxygen:

see any other information on materials and methods incl. tables

Conductivity:

see any other information on materials and methods incl. tables

Details on test conditions:

Number of daphnids 20 daphnids, divided into 4 replicates, each with 5 daphnids, were
and replicates used for each concentration level and the control.

Age of the daphnids at the start of the exposure
Less than 24 hours old daphnids from a healthy stock were used for the study. Juvenile daphnids were removed from the culture vessels at the latest 24 hours before the start of the exposure and discarded. The juveniles born within the following period of max. 24 hours preceding the exposure were used for the test. No first brood progeny was used for the test.

Acclimatization Acclimatization of the daphnids was not necessary, because the composition of the dilution water was equivalent to the culture medium.

Application 20 g test solution per replicate were weighed out into each test vessel. This corresponds to 20 mL per test vessel. The daphnids were inserted with a small amount of dilution water by a pipette.

Test temperature (target) 18 - 22 °C, constant within ± 1 °C

Illumination (target) Diffuse light, light intensity of max. 1500 lx

Photoperiod (target) 16/8 hours light/dark cycle

Feeding The daphnids were not fed during the study.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

EC10

Effect conc.:

0.563 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: CI (< 0.250 - 4.69)

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

0.261 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: (0.251 - 0.328)

Effect concentrations 3

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

3.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: CI (2.27 - 4.76)

Effect concentrations 4

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.331 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: CI(0.271 - 0.403)

Effect concentrations 5

Duration:

24 h

Dose descriptor:

EC100

Effect conc.:

> 4.83 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Effect concentrations 6

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

2.08 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Results with reference substance (positive control):

The percentage of immobility for the reference item potassium dichromate (SIGMA-ALDRICH, batch number MKBV0900V, purity 99.0%, CAS RN 7778-50-9) was determined after 24 hours from 2017-12-06 to 2017-12-07. For results of the most recent of the monthly performed reference tests, see Table 6.

EC50-Value (with 95% Confidence Limits) of the Reference Item Potassium dichromate
based on nominal concentrations mg/L, (0 - 24 hours)
Current Study Valid Range
EC50 1.64 mg/L
0.6 - 2.4 mg/L, acc. to AQS P 9/2 (02/2000); clone 5
0.6 - 2.1 mg/L, acc. to OECD 202 (2004); clone A
95% confidence limits 1.00 - 4.00 mg/L

Reported statistics and error estimates:

Methods of evaluation
The EC100 (24 and 48 hours) was empirically derived from the observed immobilization rates.
The effect levels (EC10/50/100) were based on the geometric mean measured concentrations of the test item.

EC-values and statisticalanalyses
The EC10/50-values after 24 and 48 hours were calculated by sigmoidal dose-response regression. The respective 95% confidence limits were calculated from the standard error and the t-distribution. All calculations were carried out from the best-fit values with the software GraphPad Prism. The concentration effect relationships are shown graphically.
The EC50-value for the reference item was also calculated by sigmoidal dose-response regression. The respective 95% confidence limits were calculated from the standard error and the t-distribution. All calculations were carried out from the best-fit values with the software GraphPad Prism.

Software
All data were computer-processed and rounded for presentation. Consequently, minor variations may occur from the original figures if manual calculations based on the original figures are made subsequently. Calculations were made using the following software:
- GraphPad Prism, GRAPHPAD SOFTWARE, INC.
- Excel, MICROSOFT CORPORATION

Any other information on results incl. tables

Immobilization Ratesafter 24 and 48 hours of Exposure in the Definitive Test

(n = 20, divided into 4 replicates with 5 daphnids each)

Geometric mean measured concentration [mg/L]	IMMOBILIZATION [%]
	24 hours					48 hours
	Replicates					Replicates
	1	2	3	4	MV	1	2	3	4	MV
4.83	100	100	80	100	95	100	100	100	100	100
2.08	0	0	20	40	15	100	100	100	100	100
0.964	20	40	40	20	30	80	100	100	100	95
0.476	20	0	0	0	5	100	80	60	80	80
0.250	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

Absolute Numbers of immobile daphnids after 24 and 48 hours of Exposure in the Definitive Test

             (n = 20, divided into 4 replicates with 5 daphnids each)

Geometric mean measured concentration  [mg/L]	Number of immobile Daphnids / Total number of Daphnids
	24 hours					48 hours
	Replicates					Replicates
	1	2	3	4	MV	1	2	3	4	MV
4.83	5 / 5	5 / 5	4 / 5	5 / 5	19 / 20	5 / 5	5 / 5	5 / 5	5 / 5	20 / 20
2.08	0 / 5	0 / 5	1 / 5	2 / 5	3 / 20	5 / 5	5 / 5	5 / 5	5 / 5	20 / 20
0.964	1 / 5	2 / 5	2 / 5	1 / 5	6 / 20	4 / 5	5 / 5	5 / 5	5 / 5	19 / 20
0.476	1 / 5	0 / 5	0 / 5	0 / 5	1 / 20	5 / 5	4 / 5	3 / 5	4 / 5	16 / 20
0.250	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20
Control	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20

 

 

 

Additional Observations during the Definitive Test

 

All tested concentration levels were visually clear throughout the exposure and showed a concentration-related orange coloring.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on the nominal concentrations of the test item , the 48 hour-EC50 for Daphnia magna was 0.331 (0.271 – 0.403) test item/L.

Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item were determined at the test facility according to OECD 202 (2004) from 2017-09-25 to 2017-12-15 with the definitive exposure phase from 2017-12-13 to 2017-12-15.

The study was conducted under semi-static conditions over a period of 48 hours with 5 concentrations of the test item in the range of 4.27 to 100 mg test item/L, prepared in a geometric series with a separation factor of 2.2.

All tested concentration levels were visually clear and showed a concentration-related orange coloring throughout the exposure.

Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to each concentration level and the control.

The concentrations of the test item were analytically verified via HPLC-DAD in fresh media of all concentration levels and in the control at the start of the exposure (0 hours) and in 48-hours old media at the end of the test (48 hours).

The measured concentrations of the test item in fresh media at the start of the exposure (0 hours) were in the range of < LOQ to 5.08 mg/L. The measured concentrations in the old media at the end of the test (48 hours) were in the range of 82 to 91% of the initially measured concentrations.

All effect concentrations given in the table below are based on the geometric mean measured test item concentrations of the test item .

The validity criteria of the test guidelines were fulfilled.

 

EC₁₀-, EC₅₀-and EC₁₀₀-Values

(based on the geometric mean measured concentrations of the test item)

Test Item
Effect concentrations	Test duration [hours]	Geometric mean measured test item concentrations [mg test item/L]
EC10	24	0.563 (< 0.250 – 4.69)
	48	0.261 (0.251 – 0.328)
EC50	24	3.60 (2.27 – 4.76)
	48	0.331 (0.271 – 0.403)
EC100	24	> 4.83
	48	2.08